U.S. patent application number 12/635917 was filed with the patent office on 2010-05-27 for distinguishing pca3 messenger rna species in benign and malignant prostate tissues.
Invention is credited to URSULA BUSSE, CAMILLE CHYPRE, YVES FRADET.
Application Number | 20100129824 12/635917 |
Document ID | / |
Family ID | 22560223 |
Filed Date | 2010-05-27 |
United States Patent
Application |
20100129824 |
Kind Code |
A1 |
BUSSE; URSULA ; et
al. |
May 27, 2010 |
DISTINGUISHING PCA3 MESSENGER RNA SPECIES IN BENIGN AND MALIGNANT
PROSTATE TISSUES
Abstract
This invention concerns the discovery of two distinct PCA3 mRNA
sequences. One of these sequences corresponds to a short PCA3 mRNA
molecule whereas the other PCA3 RNA molecule is longer as it
comprises an additional sequence between exon 3 and exon 4a. The
short RNA is associated with prostate cancer whereas the long RNA
sequence is associated with a non-malignant state of the prostate.
Based on the differential expression levels of these two PCA3 RNA
sequences, protocols for the diagnosis of prostate disease are
provided. The invention also relates to therapeutic approaches to
prostate cancer.
Inventors: |
BUSSE; URSULA; (SAINT-LOUIS,
FR) ; CHYPRE; CAMILLE; (ANNECY, FR) ; FRADET;
YVES; (QUEBEC, CA) |
Correspondence
Address: |
GOUDREAU GAGE DUBUC
2000 MCGILL COLLEGE, SUITE 2200
MONTREAL
QC
H3A 3H3
CA
|
Family ID: |
22560223 |
Appl. No.: |
12/635917 |
Filed: |
December 11, 2009 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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12098577 |
Apr 7, 2008 |
7655408 |
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12635917 |
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09675650 |
Sep 29, 2000 |
7368545 |
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12098577 |
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60156594 |
Sep 29, 1999 |
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Current U.S.
Class: |
435/6.14 |
Current CPC
Class: |
G01N 33/57434 20130101;
C12Q 2600/136 20130101; C07H 21/04 20130101; C07K 14/4748 20130101;
A01K 2217/05 20130101; A61P 35/00 20180101; C12Q 2600/158 20130101;
G01N 2800/56 20130101; C07K 14/47 20130101; C07K 16/3069 20130101;
A61P 13/08 20180101; G01N 2333/47 20130101; C12Q 1/6886
20130101 |
Class at
Publication: |
435/6 |
International
Class: |
C12Q 1/68 20060101
C12Q001/68 |
Foreign Application Data
Date |
Code |
Application Number |
Sep 29, 2000 |
CA |
PCT/CA00/01154 |
Claims
1. A method of determining the malignancy status of prostate cells
contained in a sample undergoing testing, said method comprising:
(a) amplifying PCA3 nucleic acids using RNA templates and a pair of
oligonucleotides which amplify across nucleotide positions 26 and
255 of SEQ ID NO:1 and across nucleotide positions 26 and 27 of SEQ
ID NO:2, thereby resulting in a collection of amplified nucleic
acids, when said PCA3 nucleic acids are present, said collection
comprising (i) a PCA3 nucleic acid that comprises an additional
sequence between PCA3 exon 3 and PCA3 exon 4a, said additional
sequence consisting essentially of nucleotides 27 to 254 of SEQ ID
NO:1; and (ii) a PCA3 nucleic acid that comprises PCA3 exon 3
joined to PCA3 exon 4a without including said additional sequence;
and (b) detecting among said collection of amplified nucleic acids
(iii) substantially only said PCA3 nucleic acid that comprises PCA3
exon 3 joined to PCA3 exon 4a without including said additional
sequence, and not said PCA3 nucleic acid that comprises said
additional sequence, if said sample comprises prostate cancer
cells; and (iv) substantially only said PCA3 nucleic acid that
comprises said additional sequence between PCA3 exon 3 and PCA3
exon 4a, and not said PCA3 nucleic acid that comprises PCA3 exon 3
joined to PCA3 exon 4a without including said additional sequence,
if prostate cells contained in the sample consist of benign
prostate cells associated with BPH, thereby determining the
malignancy status of prostate cells contained in said sample.
2. A method of distinguishing differentially expressed PCA3 nucleic
acids that indicate the malignancy status of prostate cells
contained in a sample undergoing testing, said method comprising:
(a) performing a nucleic acid amplification reaction, using RNAs
obtained from prostate cells contained in said sample as templates
to amplify PCA3 nucleic acid sequences across nucleotide positions
26 and 255 of SEQ ID NO:1, which respectively define the PCA3 exon
3-intron 3 and intron 3-exon 4a junctions, and nucleotide positions
26 and 27 of SEQ ID NO:2, which define the PCA3 exon 3-exon 4a
junction, whereby there are synthesized first and second
amplification products; wherein said first amplification product
results from an amplification of a first PCA3 RNA that comprises
portions of exon 3 and exon 4a sequences and an additional sequence
between PCA3 exon 3 and PCA3 exon 4a, said additional sequence
consisting essentially of nucleotides 27 to 254 of SEQ ID NO:1, and
wherein said second amplification product results from an
amplification of a second PCA3 RNA that comprises portions of exon
3 and exon 4a sequences with PCA3 exon 3 joined to PCA3 exon 4a
without including said additional sequence; and (b) detecting one
of said first and second amplification products independent of the
other, thereby distinguishing differentially expressed PCA3 nucleic
acids that indicate the status of prostate cells contained in said
sample.
3. The method of claim 1, wherein said pair of oligonucleotides
comprises a first oligonucleotide which binds to or upstream of
nucleotide position 26 of SEQ ID NO:1, and a second oligonucleotide
which binds to or downstream of nucleotide position 255 of SEQ ID
NO:1.
4. The method of claim 1, wherein said pair of oligonucleotides
comprises a first oligonucleotide which binds to exon 3 and a
second oligonucleotide which binds to exon 4a.
5. The method of claim 1, wherein after step (a) and before step
(b) there is an additional step of contacting said amplified
nucleic acids with a detectably labelled hybridization probe.
6. The method of claim 1, wherein said oligonucleotides are at
least 10 nucleotides in length.
7. The method of claim 1, wherein said amplifying in step (a)
comprises a reaction selected from the group consisting of a
nucleic acid sequence-based amplification reaction (NASBA), a
polymerase chain reaction (PCR), a transcription-based
amplification reaction, a strand displacement amplification (SDA),
a ligase chain reaction (LCR), and a Q.beta. replicase
reaction.
8. The method of claim 1, wherein said oligonucleotides are at
least 12 nucleotides in length.
9. The method of claim 1, wherein said oligonucleotides are at
least 15 nucleotides in length.
10. The method of claim 1, wherein said oligonucleotides are at
least 20 nucleotides in length.
11. The method of claim 1, wherein said oligonucleotides are at
least 18 to about 50 nucleotides in length.
12. The method of claim 1, wherein said oligonucleotides are at
least 20 to about 35 nucleotides in length.
13. The method of claim 5, wherein said detecting in step b)
comprises binding with a probe which hybridizes under high
stringency conditions to at least 10 consecutive nucleotides of a
sequence comprising nucleotides 26 and 27 of SEQ ID NO:2, which
define the exon 3-exon 4a junction, wherein said high stringency
conditions comprise a hybridization at 65.degree. C. in
5.times.SSC, 5.times.Denhardt's solution, 1% SDS, and 100 .mu.g/ml
denatured salmon sperm DNA.
14. The method of claim 13, wherein said probe hybridizes to at
least 15 consecutive nucleotides of said exon 3-exon 4a
junction.
15. The method of claim 13, wherein said probe hybridizes to at
least 20 consecutive nucleotides of said exon 3-exon 4a
junction.
16. The method of claim 5, wherein said probe hybridizes to at
least 15 consecutive nucleotides of said exon 3-exon 4a
junction.
17. The method of claim 5, wherein said probe hybridizes to at
least 20 consecutive nucleotides of said exon 3-exon 4a
junction.
18. The method of claim 1, wherein said pair of oligonucleotides
comprises a first oligonucleotide binding to a sequence comprised
in nucleotides 1 to 26 of SEQ ID NO:1, and a second oligonucleotide
binding to a sequence comprised in nucleotides 255 to 506 of SEQ ID
NO:1.
19. The method of claim 2, wherein said amplification is
carried-out with a pair of oligonucleotides comprising a first
oligonucleotide which binds to exon 3 and a second oligonucleotide
which binds to exon 4a.
20. A kit for determining the malignancy status of prostate cells
by distinguishing between a non-malignant PCA3 nucleic acid and a
malignant PCA3 nucleic acid in a sample undergoing testing said kit
comprising: (a) a pair of oligonucleotides which amplify across
nucleotide positions 26 and 255 of SEQ ID NO:1 and across
nucleotide positions 26 and 27 of SEQ ID NO:2, thereby resulting in
a collection of amplified nucleic acids, when said PCA3 nucleic
acids are present, said collection comprising a PCA3 nucleic acid
that comprises an additional sequence between PCA3 exon 3 and PCA3
exon 4a, said additional sequence consisting essentially of
nucleotides 27 to 254 of SEQ ID NO:1, and a PCA3 nucleic acid that
comprises PCA3 exon 3 joined to PCA3 exon 4a without including said
additional sequence; and (b) a probe hybridizing under high
stringency conditions to at least 10 consecutive nucleotides of a
nucleic acid comprising nucleotide positions 26 and 27 of SEQ ID
NO:2, which define the PCA3 exon 3-exon 4a junction, thereby
detecting among said collection of amplified nucleic acids
substantially only said PCA3 nucleic acid that comprises PCA3 exon
3 joined to PCA3 exon 4a without including said additional
sequence, and not said PCA3 nucleic acid that comprises said
additional sequence.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation application of recently
allowed U.S. application Ser. No. 12/098,577 which itself is a
divisional of U.S. patent application Ser. No. 09/675,650, now U.S.
Pat. No. 7,368,545 and which claims priority on international
Application Number PCT/CA00/01154 filed Sep. 29, 2000, which itself
claims priority on U.S. provisional application Ser. No. 60/156,594
filed Sep. 29, 1999. The patent applications identified above are
incorporated here by reference in their entirety to provide
continuity of disclosure.
SEQUENCE LISTING
[0002] This application contains a Sequence Listing in computer
readable form entitled "Sequence Listing", created Dec. 2, 2009,
having a size of 4 Ko. The computer readable form is incorporated
herein by reference.
FIELD OF THE INVENTION
[0003] The present invention relates to prostate cancer. More
specifically, the present invention relates to nucleic acid
molecules (messenger RNAs) encoded by the gene PCA3; the
differential expression of two of these RNA species in
non-malignant and malignant prostatic states; methods for
specifically diagnosing prostate cancer based on the detection of
the RNA species related to prostate cancer; therapeutic approaches
to prostate cancer implying these two RNA species; nucleic acid
molecules and antibodies having binding affinity for the
differentially expressed mRNAs; kits containing nucleic acid probes
or antibodies; bioassays using the nucleic acid sequences of the
differentially expressed mRNAs of the present invention to
diagnose, assess or prognose a mammal afflicted with or susceptible
to developing prostate cancer; and to bioassays to screen for
compounds which modulate the expression of the mRNAs of the present
invention.
BACKGROUND OF THE INVENTION
[0004] Over the last decade, cancer of the prostate has become the
most commonly diagnosed malignancy among men and the second leading
cause of male cancer deaths in the western population, following
lung cancer (Landis et al., 1998, CA Cancer J. Clin. 48(1):6-29).
Of all cancers, the incidence of prostate cancer increases most
rapidly with age. As longevity among the western population
increases, there continues to be a corresponding rise in the number
of prostate cancers with an expected increase of 60% in this decade
alone. Mortality has increased at a slower rate, but overall has
doubled in the last 50 years. Although the disease is typically
diagnosed in men over the age of 65, its impact is still
significant in that the average life span of a man who dies from
prostate cancer is reduced by 9-10 years. If discovered, early
prostate cancer can now be cured with surgery in approximately 90%
of cases. Unfortunately the disease is slowly fatal once the tumor
spreads outsize the area of the gland and forms distant metastases.
Early detection of the disease, while still confined to the
prostate gland, and accurate staging for the selection of
appropriate therapy should improve mortality rates.
[0005] Despite many advances in recent years, the precision with
which an individual suffering from prostate cancer can be staged is
still sub-optimal. The main reason for this is that tumor spread
beyond the prostate is generally microscopic rather than
macroscopic. Digital rectal examination of the prostate has been
the cornerstone for the local staging of prostatic cancer for many
decades, but it oftentimes underestimates the extent of the
disease. Transrectal ultrasound by itself is only of limited value
as a means of prostate cancer staging. Computer tomography and
magnetic resonance imaging have generally been disappointing in the
staging of prostate cancer (Kirby, 1997, Prostate cancer and
Prostatic Diseases 1:2-10). Recent promising approaches to prostate
cancer staging imply the use of biochemical and molecular
technologies, centered around proteins or their corresponding
nucleic acids which are preferentially expressed in prostate cells
(Lange, 1997, In Principles and Practice of Genitourinary Oncology
ed. Lippincott-Raven Publishers, Ch. 41, pp. 417-425). The most
notorious prostate markers are PSA (prostate specific antigen) and
PSM (prostate specific membrane) antigen.
[0006] PSA is a secreted glycoprotein encoded by the PSA gene
located on chromosome 19. It is expressed under androgen control by
glandular epithelial cells of the prostate and secreted into
seminal plasma. PSA protein is normally confined to the prostate
but in the case of prostatic disease such as cancer or BPH (benign
prostate hyperplasia), PSA leaks into the blood where it is present
in different forms, including one that is and one that is not bound
to protein complexes (EI-Shirbiny, 1994, Adv. Clin. Chem. 31:99).
The measurement of total PSA serum concentrations is one of the
most frequently used and FDA-approved biochemical tests in the
screening and management of prostate cancer patients. Studies to
date have suggested that screening with PSA, in conjunction with
digital rectal exams and transrectal ultrasound, increases the
detection of early prostate cancers often while still localized to
the gland itself (Brawer et al., 1992, J. Urol. 147:841). Serum PSA
is also useful for monitoring of patients after therapy, especially
after surgical prostatectomy. However, total PSA measurements also
identify a large number of patients with abnormally elevated levels
who are subsequently found to have no prostate cancer. Recently,
the concept of measuring the percent free/total PSA ratio was shown
to increase the specificity of prostate cancer screening in men
with PSA between 4 and 10 ng/mL (Letran et al., 1998, J. Urol.
160:426).
[0007] The PSM gene encodes a transmembrane glycoprotein expressed
by epithelial cells of normal prostate, benign prostate hyperplasia
and, to a greater extent, malignant prostatic tissue. Low levels of
PSM are also detected in some other tissues (Israeli et al., 1994,
Cancer Res. 54:1807). PSA and PSM have also been targets for
molecular approaches to prostate cancer using RT-PCR (reverse
transcription -polymerase chain reaction). This very sensitive
nucleic acid amplification technology is used to identify cells
based on the expression of specific messenger RNAs. It involves
preparing RNA samples from tissues or body fluids, reverse
transcribing it into cDNA and amplifying specific cDNAs by the use
of primers that target the particular gene of interest. RT-PCR
analyses of blood, lymph nodes and bone marrow from prostate cancer
patients using PSA and PSM have disclosed the extreme sensitivity
of this approach. However, the clinical value of molecular tests
still has to be confirmed (Verkaik et al., 1997, Urol. Res. 25:373;
Gomella et al., 1997, J. Urol. 158:326).
[0008] Thus, there remains a need to provide a more sensitive test
for diagnosing prostate cancer. There also remains a need to
provide a better test for the staging of prostate cancer. There
also remains a need to provide a prostate cancer marker which is
more specific and more reliable to prostate cancer detection,
staging and treatment methods.
[0009] The present invention seeks to meet these and other
needs.
[0010] A new prostate cancer marker, PCA3, was discovered a few
years ago by differential display analysis intended to highlight
genes associated with prostate cancer development (PCT application
number PCT/CA98/00346). PCA3 is located on chromosome 9 and
composed of four exons. It encodes at least four different
transcripts which are generated by alternative splicing and
polyadenylation. By RT-PCR analysis, PCA3 expression was found to
be limited to the prostate and absent in all other tissues,
including testis, ovary, breast and bladder. Northern blot analysis
showed that PCA3 is highly expressed in the vast majority of
prostate cancers examined (47 out of 50) whereas no or very low
expression is detected in benign prostate hyperplasia or normal
prostate cells from the same patients. There is at least 20-fold
overexpression of PCA3 in prostatic carcinomas in comparison to
normal or BPH tissues. PCA3 expression seems to increase with tumor
grade and is detected in metastatic lesions.
[0011] In summary, prostate cancer staging based on specific
markers such as PSA and PSM is a very promising avenue for the
management of the disease. The drawback of using PSA or PSM for
prostate cancer staging is that they are expressed in normal as
well as in cancerous cells. In addition, poorly differentiated
tumors may escape diagnosis since they tend to produce
significantly less PSA protein than less aggressive tumors. This is
the case for 10% of all prostate cancers. PCA3, on the other hand,
is differentially expressed in cancerous and normal prostate cells,
and its expression does not decrease with increasing tumor grade.
PCA3 could therefore be a useful tool which may overcome the
drawbacks of PSA and PSM in the diagnosis, staging and treatment of
prostate cancer patients.
[0012] The present description refers to a number of documents, the
content of which is herein incorporated by reference, in their
entirety.
SUMMARY OF THE INVENTION
[0013] The invention concerns the discovery of distinct PCA3 RNAs
associated with a non-malignant and/or malignant status of the
prostate.
[0014] The invention also concerns the identification that a
balance between the level of these PCA3 mRNAs correlates with the
non-malignant or the malignant status of the prostate.
[0015] One of these RNAs corresponds to a PCA3 RNA molecule having
an additional sequence of 228 by (shown in SEQ ID NO:1), inserted
between exons 3 and 4a, whereas the other lacks the additional
sequence (SEQ ID NO:2). The RNA lacking the additional sequence is
associated with prostate cancer whereas the RNA comprising same is
associated with a non-malignant prostatic state. Based on the
differential expression of these two PCA3 RNA species, protocols
for the diagnosis of prostate disease are provived. The above
findings could also lead to a therapeutic approach to prostate
cancer.
[0016] The invention further concerns reagents and methods to
assess the prostate status in an animal, comprising a quantitative
determination of SEQ ID NO:1 or fragments, or variants thereof with
respect to SEQ ID NO:2 or fragments, or variants thereof.
[0017] Thus, the present invention relates to the discovery and
characterization of a novel sequence expressed in PCA3 mRNA, which
enables a determination of the prostate status of an animal, based
on a determination of the relative abundance of two differentially
expressed PCA3 mRNAs.
[0018] The invention provides, in general, isolated nucleic acid
molecules encoding differentially expressed PCA3 mRNAs and to
variants or portions thereof, retaining their ability to enable a
prostate status determination.
[0019] The invention further provides purified polypeptides encoded
by the differentially expressed PCA3 mRNAs of the present invention
or an epitope binding portion thereof.
[0020] The invention also provides nucleic acids for the specific
detection of the presence of differentially expressed PCA3 mRNAs
associated with prostate cancer or proteins or polypeptides encoded
by such mRNAs in a sample.
[0021] The invention further provides a method of detecting nucleic
acid encoding differentially expressed PCA3 mRNAs.
[0022] The invention also provides a kit for detecting the presence
of nucleic acid encoding differentially-expressed PCA3 mRNAs in a
sample.
[0023] The invention in addition provides a recombinant nucleic
acid molecule comprising, 5' to 3', a promoter effective to
initiate transcription in a host cell and the above-described
isolated nucleic acid molecule, variants or fragments thereof,
encoding differentially expressed PCA3 mRNAs.
[0024] The invention also provides a recombinant nucleic acid
molecule comprising a vector and the above-described isolated
nucleic acid molecule encoding differentially expressed PCA3
mRNAs.
[0025] The invention further provides an antisense nucleic acid
molecule specific to the differentially expressed PCA3 mRNAs.
[0026] The invention also provides a cell that contains an
above-described recombinant nucleic acid molecule.
[0027] The invention further relates to a non-human organism that
contains an above-described recombinant nucleic acid molecule
encoding a differentially expressed PCA3 mRNA. In particular, the
invention relates to a non-human organism containing a recombinant
nucleic acid molecule encoding a PCA3 mRNA having an additional
sequence between exon 3 and exon 4a. In a particularly preferred
embodiment, this additional sequence comprises the sequence of SEQ
ID NO:1, variants of parts thereof.
[0028] The invention also relates to an antibody having binding
affinity specifically to a polypeptide encoded by a differentially
expressed PCA3 mRNA or an epitope-bearing portion thereof.
[0029] The invention further provides a method of detecting
differentially expressed PCA3 mRNAs in a sample. As well, it also
provides a method of measuring the amount of differentially
expressed PCA3 mRNAs in a sample.
[0030] The invention further relates to a method of detecting
antibodies having binding affinity specifically to polypeptides
encoded by a differentially expressed PCA3 mRNA.
[0031] In one embodiment, the invention further relates to a
diagnostic kit comprising a first container means containing
nucleic acid molecules specific for a differentially expressed PCA3
mRNA, and a second container means containing a probe specific to a
differentially expressed PCA3 mRNAs.
[0032] In another embodiment, the invention relates to a diagnostic
kit comprising a first container means containing the
above-described antibody, and a second container means containing a
conjugate comprising a binding partner of the monoclonal antibody
and a label.
[0033] The invention also provides a hybridoma which produces the
above-described monoclonal antibody.
[0034] As well, the invention further relates to diagnostic methods
for human disease, in particular, prostate cancer. Preferably, a
method of diagnosing the presence or predisposition to develop
prostate cancer in a patient is provided herein.
[0035] The invention also provides methods for therapeutic uses
involving all or part of (1) a nucleic acid sequence encoding the
differentially expressed PCA3 mRNAs, variants or parts thereof, (2)
antisense to differentially expressed PCA3 mRNA molecules, variants
or parts thereof, (3) protein encoded by a differentially expressed
PCA3 mRNA, variants or parts thereof, or (4) antibodies to proteins
encoded by differentially expressed PCA3 mRNAs.
[0036] Further, the invention provides a method to modulate the
level of differentially expressed of a first PCA3 mRNA (e.g. the
long one) and a second PCA3 mRNA (e.g. the short one) by expressing
one of the first or second differentially expressed mRNA. In a
preferred embodiment, the invention provides a modulation of the
differentially expressed PCA3 mRNAs such that the level of the
first PCA3 mRNA is superior to that of the second.
[0037] Having identified the differential expression of mRNAs as a
marker for prostatic state of an animal, and more particularly
having shown that the presence of the additional sequence, which
interrupts the coding sequence of the PCA3 encoded protein,
correlates with a non-malignant state, while the absence of the
additional sequence and a non-interruption of the thereby encoded
protein, correlates with malignant cancer, the present invention
therefore provides the means to interrupt the coding sequence of
the PCA3 protein, using any means of genetic engineering, known to
a skilled artisan, and assesses whether such an interruption can
revert the malignant phenotype.
[0038] In order to provide a clear and consistent understanding of
terms used in the present description, a number of definitions are
provided hereinbelow.
[0039] As used herein, the terminology "non-malignant prostate or
status" is meant to cover a non-cancerous prostatic state. Thus,
these terminologies are meant to include a normal status as well as
a benign prostatic status (such as BPH, for example).
[0040] Since the differentiating markers between the malignant and
non-malignant prostatic state is at the mRNA and protein level
(i.e. an expressed marker), one of the advantages of the present
invention is to enable a determination of the prostatic status in
an animal using a number of available means to the skilled artisan.
Non-limiting examples of such means include nucleic acid probes,
antibodies, ligands and PNAs, in easily obtainable cells which
express these differentiating markers. A non-limiting example
thereof is lymphocytes, thereby enabling a determination from a
simple blood sample.
[0041] The term "sample" is used herein broadly to refer to all
types of samples from an animal in which the differential
expression of the short and/or long PCA3 nucleic acid or protein of
the present invention can be analyzed. Non-limiting examples
thereof include biopsies, blood, fine needle aspirate, urine and
bone marrow.
[0042] Nucleotide sequences are presented herein by single strand,
in the 5' to 3' direction, from left to right, using the one letter
nucleotide symbols as commonly used in the art and in accordance
with the recommendations of the IUPAC-IUB Biochemical Nomenclature
Commission.
[0043] Unless defined otherwise, the scientific and technological
terms and nomenclature used herein have the same meaning as
commonly understood by a person of ordinary skill to which this
invention pertains. Generally, the procedures for cell cultures,
infection, molecular biology methods and the like are common
methods used in the art. Such standard techniques can be found in
reference manuals such as for example Sambrook et al. (1989,
Molecular Cloning--A Laboratory Manual, Cold Spring Harbor
Laboratories) and Ausubel et al. (1994, Current Protocols in
Molecular Biology, Wiley, New York).
[0044] The present description refers to a number of routinely used
recombinant DNA (rDNA) technology terms. Nevertheless, definitions
of selected examples of such rDNA terms are provided for clarity
and consistency.
[0045] As used herein, "nucleic acid molecule", refers to a polymer
of nucleotides. Non-limiting examples thereof include DNA (i.e.
genomic DNA, cDNA) and RNA molecules (i.e. mRNA). The nucleic acid
molecule can be obtained by cloning techniques or synthesized. DNA
can be double-stranded or single-stranded (coding strand or
non-coding strand [antisense]).
[0046] The term "recombinant DNA" as known in the art refers to a
DNA molecule resulting from the joining of DNA segments. This is
often referred to as genetic engineering.
[0047] The term "DNA segment", is used herein, to refer to a DNA
molecule comprising a linear stretch or sequence of nucleotides.
This sequence when read in accordance with the genetic code, can
encode a linear stretch or sequence of amino acids which can be
referred to as a polypeptide, protein, protein fragment and the
like.
[0048] The terminology "amplification pair" refers herein to a pair
of oligonucleotides (oligos) of the present invention, which are
selected to be used together in amplifying a selected nucleic acid
sequence by one of a number of types of amplification processes,
preferably a polymerase chain reaction. Other types of
amplification processes include ligase chain reaction, strand
displacement amplification, or nucleic acid sequence-based
amplification, as explained in greater detail below. As commonly
known in the art, the oligos are designed to bind to a
complementary sequence under selected conditions.
[0049] In one particular embodiment, amplification of a nucleic
acid sample from a patient is amplified under conditions which
favor the amplification of the most abundant differentially
expressed nucleic acid. In one preferred embodiment, RT-PCR is
carried out on an mRNA sample from a patient under conditions which
favor the amplification of the most abundant PCA3 mRNA. In another
preferred embodiment, the amplification of the differentially
expressed PCA3 nucleic acids is carried out simultaneously. Of
course, it will be realized by a person skilled in the art that
such methods could be adapted for the detection of differentially
expressed proteins instead of differentially expressed nucleic acid
sequences.
[0050] The nucleic acid (i.e. DNA or RNA) for practicing the
present invention may be obtained according to well known
methods.
[0051] Oligonucleotide probes or primers of the present invention
may be of any suitable length, depending on the particular assay
format and the particular needs and targeted genomes employed. In
general, the oligonucleotide probes or primers are at least 12
nucleotides in length, preferably between 15 and 24 molecules, and
they may be adapted to be especially suited to a chosen nucleic
acid amplification system. As commonly known in the art, the
oligonucleotide probes and primers can be designed by taking into
consideration the melting point of hydrizidation thereof with its
targeted sequence (see below and in Sambrook et al., 1989,
Molecular Cloning--A Laboratory Manual, 2nd Edition, CSH
Laboratories; Ausubel et al., 1989, in Current Protocols in
Molecular Biology, John Wiley & Sons Inc., N.Y.).
[0052] The term "oligonucleotide" or DNA'' molecule or sequence
refers to a molecule comprised of the deoxyribonucleotides adenine
(A), guanine (G), thymine (T) and/or cytosine (C). When in a
double-stranded form, it can comprise or include a "regulatory
element" according to the present invention, as the term is defined
herein. The term "oligonucleotide" or "DNA" can be found in linear
DNA molecules or fragments, viruses, plasmids, vectors, chromosomes
or synthetically derived DNA. As used herein, particular
double-stranded DNA sequences may be described according to the
normal convention of giving only the sequence in the 5' to 3'
direction. It will also be recognized that "oligonucleotide" can be
in a single-stranded form.
[0053] "Nucleic acid hybridization" refers generally to the
hybridization of two single-stranded nucleic acid molecules having
complementary base sequences, which under appropriate conditions
will form a thermodynamically favored double-stranded structure.
Examples of hybridization conditions can be found in the two
laboratory manuals referred above (Sambrook et al., 1989, supra and
Ausubel et al., 1989, supra) and are commonly known in the art. In
the case of a hybridization to a nitrocellulose filter, as for
example in the well known Southern blotting procedure, a
nitrocellulose filter can be incubated overnight at 65.degree. C.
with a labeled probe in a solution containing 50% formamide, high
salt (5.times.SSC or 5.times.SSPE), 5.times.Denhardt's solution, 1%
SDS, and 100 .mu.g/ml denatured carrier DNA (i.e. salmon sperm
DNA). The non-specifically binding probe can then be washed off the
filter by several washes in 0.2.times.SSC/0.1% SDS at a temperature
which is selected in view of the desired stringency: room
temperature (low stringency), 42.degree. C. (moderate stringency)
or 65.degree. C. (high stringency). The selected temperature is
based on the melting temperature (Tm) of the DNA hybrid. Of course,
RNA-DNA hybrids can also be formed and detected. In such cases, the
conditions of hybridization and washing can be adapted according to
well known methods by the person of ordinary skill. Stringent
conditions will be preferably used (Sambrook et al., 1989,
supra).
[0054] Probes of the invention can be utilized with naturally
occurring sugar-phosphate backbones as well as modified backbones
including phosphorothioates, dithionates, alkyl phosphonates and
.alpha.-nucleotides and the like. Modified sugar-phosphate
backbones are generally taught by Miller, 1988, Ann. Reports Med.
Chem. 23:295 and Moran et al., 1987, Nucleic acid molecule. Acids
Res., 14:5019. Probes of the invention can be constructed of either
ribonucleic acid (RNA) or deoxyribonucleic acid (DNA), and
preferably of DNA.
[0055] The types of detection methods in which probes can be used
include Southern blots (DNA detection), dot or slot blots (DNA,
RNA), and Northern blots (RNA detection). Although less preferred,
labeled proteins could also be used to detect a particular nucleic
acid sequence to which it binds. More recently, PNAs have been
described (Nielsen et al. 1999, Current Opin. Biotechnol.
10:71-75). PNAs could also be used to detect the mRNAs of the
present invention. Other detection methods include kits containing
probes on a dipstick setup and the like.
[0056] Although the present invention is not specifically dependent
on the use of a label for the detection of a particular nucleic
acid sequence, such a label might be beneficial, by increasing the
sensitivity of the detection. Furthermore, it enables automation.
Probes can be labeled according to numerous well known methods
(Sambrook et al., 1989, supra). Non-limiting examples of labels
include .sup.3H, .sup.14C, .sup.32P, and .sup.35S. Non-limiting
examples of detectable markers include ligands, fluorophores,
chemiluminescent agents, enzymes, and antibodies. Other detectable
markers for use with probes, which can enable an increase in
sensitivity of the method of the invention, include biotin and
radionucleotides. It will become evident to the person of ordinary
skill that the choice of a particular label dictates the manner in
which it is bound to the probe.
[0057] As commonly known, radioactive nucleotides can be
incorporated into probes of the invention by several methods.
Non-limiting examples thereof include kinasing the 5' ends of the
probes using gamma .sup.32P ATP and polynucleotide kinase, using
the Klenow fragment of Pol I of E. coli in the presence of
radioactive dNTP (i.e. uniformly labeled DNA probe using random
oligonucleotide primers in low-melt gels), using the SP6/T7 system
to transcribe a DNA segment in the presence of one or more
radioactive NTP, and the like.
[0058] As used herein, "oligonucleotides" or "oligos" define a
molecule having two or more nucleotides (ribo or
deoxyribonucleotides). The size of the oligo will be dictated by
the particular situation and ultimately on the particular use
thereof and adapted accordingly by the person of ordinary skill. An
oligonucleotide can be synthetised chemically or derived by cloning
according to well known methods.
[0059] As used herein, a "primer" defines an oligonucleotide which
is capable of annealing to a target sequence, thereby creating a
double stranded region which can serve as an initiation point for
DNA synthesis under suitable conditions.
[0060] Amplification of a selected, or target, nucleic acid
sequence may be carried out by a number of suitable methods. See
generally Kwoh et al., 1990, Am. Biotechnol. Lab. 8:14-25. Numerous
amplification techniques have been described and can be readily
adapted to suit particular needs of a person of ordinary skill.
Non-limiting examples of amplification techniques include
polymerase chain reaction (PCR), ligase chain reaction (LCR),
strand displacement amplification (SDA), transcription-based
amplification, the Q.beta. replicase system and NASBA (Kwoh et al.,
1989, Proc. Natl. Acad. Sci. USA 86, 1173-1177; Lizardi et al.,
1988, BioTechnology 6:1197-1202; Malek et al., 1994, Methods Mol.
Biol., 28:253-260; and Sambrook et al., 1989, supra). Preferably,
amplification will be carried out using PCR.
[0061] Polymerase chain reaction (PCR) is carried out in accordance
with known techniques. See, e.g., U.S. Pat. Nos. 4,683,195;
4,683,202; 4,800,159; and 4,965,188 (the disclosures of all three
U.S. Patent are incorporated herein by reference). In general, PCR
involves, a treatment of a nucleic acid sample (e.g., in the
presence of a heat stable DNA polymerase) under hybridizing
conditions, with one oligonucleotide primer for each strand of the
specific sequence to be detected. An extension product of each
primer which is synthesized is complementary to each of the two
nucleic acid strands, with the primers sufficiently complementary
to each strand of the specific sequence to hybridize therewith. The
extension product synthesized from each primer can also serve as a
template for further synthesis of extension products using the same
primers. Following a sufficient number of rounds of synthesis of
extension products, the sample is analysed to assess whether the
sequence or sequences to be detected are present. Detection of the
amplified sequence may be carried out by visualization following
EtBr staining of the DNA following gel electrophores, or using a
detectable label in accordance with known techniques, and the like.
Fora review on PCR techniques (see PCR Protocols, A Guide to
Methods and Amplifications, Michael et al. Eds, Acad. Press,
1990).
[0062] Ligase chain reaction (LCR) is carried out in accordance
with known techniques (Weiss, 1991, Science 254:1292). Adaptation
of the protocol to meet the desired needs can be carried out by a
person of ordinary skill. Strand displacement amplification (SDA)
is also carried out in accordance with known techniques or
adaptations thereof to meet the particular needs (Walker et al.,
1992, Proc. Natl. Acad. Sci. USA 89:392-396; and ibid., 1992,
Nucleic Acids Res. 20:1691-1696).
[0063] As used herein, the term "gene" is well known in the art and
relates to a nucleic acid sequence defining a single protein or
polypeptide. A "structural gene" defines a DNA sequence which is
transcribed into RNA and translated into a protein having a
specific amino acid sequence thereby giving rise the a specific
polypeptide or protein. It will be readily recognized by the person
of ordinary skill, that the nucleic acid sequence of the present
invention can be incorporated into anyone of numerous established
kit formats which are well known in the art.
[0064] A "heterologous" (i.e. a heterologous gene) region of a DNA
molecule is a subsegment segment of DNA within a larger segment
that is not found in association therewith in nature. The term
"heterologous" can be similarly used to define two polypeptidic
segments not joined together in nature. Non-limiting examples of
heterologous genes include reporter genes such as luciferase,
chloramphenicol acetyl transferase, -galactosidase, and the like
which can be juxtaposed or joined to heterologous control regions
or to heterologous polypeptides.
[0065] The term "vector" is commonly known in the art and defines a
plasmid DNA, phage DNA, viral DNA and the like, which can serve as
a DNA vehicle into which DNA of the present invention can be
cloned. Numerous types of vectors exist and are well known in the
art.
[0066] The term "expression" defines the process by which a gene is
transcribed into mRNA (transcription), the mRNA is then being
translated (translation) into one polypeptide (or protein) or
more.
[0067] The terminology "expression vector" defines a vector or
vehicle as described above but designed to enable the expression of
an inserted sequence following transformation into a host. The
cloned gene (inserted sequence) is usually placed under the control
of control element sequences such as promoter sequences. The
placing of a cloned gene under such control sequences is often
refered to as being operably linked to control elements or
sequences.
[0068] Operably linked sequences may also include two segments that
are transcribed onto the same RNA transcript. Thus, two sequences,
such as a promoter and a "reporter sequence" are operably linked if
transcription commencing in the promoter will produce an RNA
transcript of the reporter sequence. In order to be "operably
linked" it is not necessary that two sequences be immediately
adjacent to one another.
[0069] Expression control sequences will vary depending on whether
the vector is designed to express the operably linked gene in a
prokaryotic or eukaryotic host or both (shuttle vectors) and can
additionally contain transcriptional elements such as enhancer
elements, termination sequences, tissue-specificity elements,
and/or translational initiation and termination sites.
[0070] Prokaryotic expressions are useful for the preparation of
large quantities of the protein encoded by the DNA sequence of
interest. This protein can be purified according to standard
protocols that take advantage of the intrinsic properties thereof,
such as size and charge (i.e. SDS gel electrophoresis, gel
filtration, centrifugation, ion exchange chromatography . . . ). In
addition, the protein of interest can be purified via affinity
chromatography using polyclonal or monoclonal antibodies. The
purified protein can be used for therapeutic applications.
[0071] The DNA construct can be a vector comprising a promoter that
is operably linked to an oligonucleotide sequence of the present
invention, which is in turn, operably linked to a heterologous
gene, such as the gene for the luciferase reporter molecule.
"Promoter" refers to a DNA regulatory region capable of binding
directly or indirectly to RNA polymerase in a cell and initiating
transcription of a downstream (3' direction) coding sequence. For
purposes of the present invention, the promoter is bound at its 3'
terminus by the transcription initiation site and extends upstream
(5' direction) to include the minimum number of bases or elements
necessary to initiate transcription at levels detectable above
background. Within the promoter will be found a transcription
initiation site (conveniently defined by mapping with S1 nuclease),
as well as protein binding domains (consensus sequences)
responsible for the binding of RNA polymerase. Eukaryotic promoters
will often, but not always, contain "TATA" boses and "CCAT" boxes.
Prokaryotic promoters contain -10 and -35 consensus sequences,
which serve to initiate transcription and the transcript products
contain a Shine-Dalgarno sequence, which serves a ribosome binding
sequences during translation initiation.
[0072] As used herein, the designation "functional derivative"
denotes, in the context of a functional derivative of a sequence
whether an nucleic acid or amino acid sequence, a molecule that
retains a biological activity (either function or structural) that
is substantially similar to that of the original sequence. This
functional derivative or equivalent may be a natural derivative or
may be prepared synthetically. Such derivatives include amino acid
sequences having substitutions, deletions, or additions of one or
more amino acids, provided that the biological activity of the
protein is conserved. The same applies to derivatives of nucleic
acid sequences which can have substitutions, deletions, or
additions of one or more nucleotides, provided that the biological
activity of the sequence is generally maintained. When relating to
a protein sequence, the substituting amino acid as chemico-physical
properties which are similar to that of the substituted amino acid.
The similar chemico-physical properties include, similarities in
charge, bulkiness, hydrophobicity, hydrophylicity and the like. The
term "functional derivatives" is intended to include "fragments",
"segments", "variants", "analogs" or "chemical derivatives" of the
subject matter of the present invention.
[0073] Thus, the term "variant" refers herein to a protein or
nucleic acid molecule which is substantially similar in structure
and biological activity to the protein or nucleic acid of the
present invention.
[0074] The functional derivatives of the present invention can be
synthesized chemically or produced through recombinant DNA
technology. All these methods are well known in the art.
[0075] As used herein, "chemical derivatives" is meant to cover
additional chemical moieties not normally part of the subject
matter of the invention. Such moieties could affect the
physico-chemical characteristic of the derivative (i.e. solubility,
absorption, half life and the like, decrease of toxicity). Such
moieties are examplified in Remington's Pharmaceutical Sciences
(1980). Methods of coupling these chemical-physical moieties to a
polypeptide are well known in the art.
[0076] The term "allele" defines an alternative form of a gene
which occupies a given locus on a chromosome.
[0077] As commonly known, a "mutation" is a detectable change in
the genetic material which can be transmitted to a daughter cell.
As well known, a mutation can be, for example, a detectable change
in one or more deoxyribonucleotide. For example, nucleotides can be
added, deleted, substituted for, inverted, or transposed to a new
position. Spontaneous mutations and experimentally induced
mutations exist. The result of a mutations of nucleic acid molecule
is a mutant nucleic acid molecule. A mutant polypeptide can be
encoded from this mutant nucleic acid molecule.
[0078] As used herein, the term purified refers to a molecule
having been separated from a cellular component. Thus, for example,
a purified protein has been purified to a level not found in
nature. A substantially pure molecule is a molecule that is lacking
in all other cellular components.
[0079] As used herein, the terms molecule, compound, or agent are
used interchangeably and broadly to refer to natural, synthetic or
semi-synthetic molecules or compounds. The term molecule therefore
denotes for example chemicals, macromolecules, cell or tissue
extracts (from plants or animals) and the like. Non limiting
examples of molecules include nucleic acid molecules, peptides,
ligands, including antibodies, carbohydrates and pharmaceutical
agents. The agents can be selected and screened by a variety of
means including random screening, rational selection and by
rational design using for example protein or ligand modelling
methods such as computer modelling. The terms rationally selected
or rationally designed are meant to define compounds which have
been chosen based on the configuration of the interaction domains
of the present invention. As will be understood by the person of
ordinary skill, macromolecules having non-naturally occurring
modifications are also within the scope of the term molecule. For
example, peptidomimetics, well known in the pharmaceutical industry
and generally referred to as peptide analogs can be generated by
modelling as mentioned above. Similarly, in a preferred embodiment,
the polypeptides of the present invention are modified to enhance
their stability. It should be understood that in most cases this
modification should not alter the biological activity of the
protein. The molecules identified in accordance with the teachings
of the present invention have a therapeutic value in diseases or
conditions in which the physiology or homeastasis of the cell
and/or tissue is compromised by a defect in the expression of PCA3
mRNAs. Alternatively, the molecules identified in accordance with
the teachings of the present invention find utility in the
development of compounds which can modulate the expression of a
differentially expressed PCA3 mRNA or modulate the activity or
level of a protein encoded thereby.
[0080] As used herein, agonists and antagonists also include
potentiators of known compounds with such agonist or antagonist
properties. In one embodiment, modulators of the level or the
activity of the PCA3 protein lacking the additional sequence of the
present invention can be identified and selected by contacting the
indicator cell with a compound or mixture or library of molecules
for a fixed period of time. In certain embodiments, the additional
sequence-containing PCA3 protein can serve as a control.
[0081] The present invention also provides antisense nucleic acid
molecules which can be used for example to decrease or abrogate the
expression of the PCA3 mRNA lacking the additional sequence of the
present invention or of the protein encoded thereby. An antisense
nucleic acid molecule according to the present invention refers to
a molecule capable of forming a stable duplex or triplex with a
portion of its targeted nucleic acid sequence (DNA or RNA). The use
of antisense nucleic acid molecules and the design and modification
of such molecules is well known in the art as described for example
in WO 96/32966, WO 96/11266, WO 94/15646, WO 93/08845 and U.S. Pat.
No. 5,593,974. Antisense nucleic acid molecules according to the
present invention can be derived from the nucleic acid sequences
and modified in accordance to well known methods. For example, some
antisense molecules can be designed to be more resistant to
degradation to increase their affinity to their targeted sequence,
to affect their transport to chosen cell types or cell
compartments, and/or to enhance their lipid solubility by using
nucleotide analogs and/or substituting chosen chemical fragments
thereof, as commonly known in the art.
[0082] An indicator cell in accordance with the present invention
can be used to identify antagonists. For example, the test molecule
or molecules are incubated with the host cell in conjunction with
one or more agonists held at a fixed concentration. An indication
and relative strength of the antagonistic properties of the
molecule(s) can be provided by comparing the level of gene
expression in the indicator cell in the presence of the agonist, in
the absence of test molecules vs in the presence thereof. Of
course, the antagonistic effect of a molecule can also be
determined in the absence of agonist, simply by comparing the level
of expression of the reporter gene product in the presence and
absence of the test molecule(s).
[0083] It shall be understood that the "in vivo" experimental model
can also be used to carry out an "in vitro" assay. For example,
cellular extracts from the indicator cells can be prepared and used
in one of the aforementioned "in vitro" tests.
[0084] As used herein the recitation indicator cells refers to
cells that express a differentially expressed PCA3 mRNA according
to the present invention. In some embodiment, the protein encoded
by the nucleic acid sequence can be coupled to an identifiable or
selectable phenotype or characteristic. Such indicator cells can be
used in the screening assays of the present invention. In certain
embodiments, the indicator cells have been engineered so as to
express a chosen derivative, fragment, homolog, or mutant of the
differentially-expressed PCA3 mRNA of the present invention. The
cells can be yeast cells or higher eukaryotic cells such as
mammalian cells. When the binding partner for the PCA3 proetin will
have been identified, the interaction between the two partners will
be able to serve as a target for the modulation of the activity of
this PCA3-encoded protein. In one particular embodiment, the
indicator cell would be a yeast cell harboring vectors enabling the
use of the two hybrid system technology, as well known in the art
(Ausubel et al., 1994, supra) and can be used to test a compound or
a library thereof. In one embodiment, a reporter gene encoding a
selectable marker or an assayable protein can be operably linked to
a control element such that expression of the selectable marker or
assayable protein is dependent on the interaction of the
PCA3-encoded protein and its binding partner. Such an indicator
cell could be used to rapidly screen at high-throughput a vast
array of test molecules. In a particular embodiment, the reporter
gene is luciferase or .beta.-Gal.
[0085] In some embodiments, it might be beneficial to express a
protein of the present invention as a fusion protein. The design of
constructs therefor and the expression and production of fusion
proteins and are well known in the art (Sambrook et al., 1989,
supra; and Ausubel et al., 1994, supra).
[0086] Non limiting examples of such fusion proteins include a
hemaglutinin fusions and Gluthione-S-transferase (GST) fusions and
Maltose binding protein (MBP) fusions. In certain embodiments, it
might be beneficial to introduce a protease cleavage site between
the two polypeptide sequences which have been fused. Such protease
cleavage sites between two heterologously fused polypeptides are
well known in the art.
[0087] In certain embodiments, it might also be beneficial to fuse
the protein of the present invention to signal peptide sequences
enabling a secretion of the fusion protein from the host cell.
Signal peptides from diverse organisms are well known in the art.
Bacterial OmpA and yeast Suc2 are two non limiting examples of
proteins containing signal sequences. In certain embodiments, it
might also be beneficial to introduce a linker (commonly known)
between the interaction domain and the heterologous polypeptide
portion. Such fusion protein find utility in the assays of the
present invention as well as for purification purposes, detection
purposes and the like.
[0088] For certainty, the sequences and polypeptides useful to
practice the invention include without being limited thereto
mutants, homologs, subtypes, alleles and the like. It shall be
understood that generally, the sequences of the present invention
should encode a functional (albeit defective) PCA3 protein. It will
be clear to the person of ordinary skill that whether the PCA3 of
the present invention, variant, derivative, or fragment thereof
retains its can be determined by using the teachings and assays of
the present invention and the general teachings of the art.
[0089] As exemplified herein below, the PCA3 protein of the present
invention can be modified, for example by in vitro mutagenesis, to
dissect the structure-function relationship thereof and permit a
better design and identification of modulating compounds. However,
some derivative or analogs having lost their biological function
may still find utility, for example for raising antibodies. These
antibodies could be used for detection or purification purposes. In
addition, these antibodies could also act as competitive or
non-competitive inhibitor and be found to be modulators of the
activity of the PCA3 protein of the present invention.
[0090] A host cell or indicator cell has been "transfected by
exogenous or heterologous DNA (e.g. a DNA construct) when such DNA
has been introduced inside the cell. The transfecting DNA may or
may not be integrated (covalently linked) into chromosomal DNA
making up the genome of the cell. In prokaryotes, yeast, and
mammalian cells for example, the transfecting DNA may be maintained
on a episomal element such as a plasmid. With respect to eukaryotic
cells, a stably transfected cell is one in which the transfecting
DNA has become integrated into a chromosome so that it is inherited
by daughter cells through chromosome replication. This stability is
demonstrated by the ability of the eukaryotic cell to establish
cell lines or clones comprised of a population of daughter cells
containing the transfecting DNA. Transfection methods are well
known in the art (Sambrook et al., 1989, supra; Ausubel et al.,
1994 supra). The use of a mammalian cell as indicator can provide
the advantage of furnishing an intermediate factor, which permits
for example the interaction of two polypeptides which are tested,
that might not be present in lower eukaryotes or prokaryotes. It
will be understood that extracts from mammalian cells for example
could be used in certain embodiments, to compensate for the lack of
certain factors.
[0091] In general, techniques for preparing antibodies (including
monoclonal antibodies and hybridomas) and for detecting antigens
using antibodies are well known in the art (Campbell, 1984, In
Monoclonal Antibody Technology: Laboratory Techniques in
Biochemistry and Molecular Biology, Elsevier Science Publisher,
Amsterdam, The Netherlands) and in Harlow et al., 1988 (in:
Antibody-A Laboratory Manual, CSH Laboratories). The present
invention also provides polyclonal, monoclonal antibodies, or
humanized versions thereof, chimeric antibodies and the like which
inhibit or neutralize their respective interaction domains and/or
are specific thereto.
[0092] From the specification and appended claims, the term
therapeutic agent should be taken in a broad sense so as to also
include a combination of at least two such therapeutic agents.
Further, the DNA segments or proteins according to the present
invention can be introduced into individuals in a number of ways.
For example, prostatic cells can be isolated from the afflicted
individual, transformed with a DNA construct according to the
invention and reintroduced to the afflicted individual in a number
of ways. Alternatively, the DNA construct can be administered
directly to the afflicted individual. The DNA construct can also be
delivered through a vehicle such as a liposome, which can be
designed to be targeted to a specific cell type, and engineered to
be administered through different routes.
[0093] For administration to humans, the prescribing medical
professional will ultimately determine the appropriate form and
dosage for a given patient, and this can be expected to vary
according to the chosen therapeutic regimen (i.e. DNA construct,
protein, cells), the response and condition of the patient as well
as the severity of the disease.
[0094] Composition within the scope of the present invention should
contain the active agent (i.e. fusion protein, nucleic acid, and
molecule) in an amount effective to achieve the desired therapeutic
effect while avoiding adverse side effects. Typically, the nucleic
acids in accordance with the present invention can be administered
to mammals (i.e. humans) in doses ranging from 0.005 to 1 mg per kg
of body weight per day of the mammal which is treated.
Pharmaceutically acceptable preparations and salts of the active
agent are within the scope of the present invention and are well
known in the art (Remington's Pharmaceutical Science, 16th Ed.,
Mack Ed.). For the administration of polypeptides, antagonists,
agonists and the like, the amount administered should be chosen so
as to avoid adverse side effects. The dosage will be adapted by the
clinician in accordance with conventional factors such as the
extent of the disease and different parameters from the patient.
Typically, 0.001 to 50 mg/kg/day will be administered to the
mammal.
[0095] The present invention relates to a kit for diagnosing and/or
staging prostate cancer or a predisposition to contracting same
comprising a nucleic acid, a protein or a ligand in accordance with
the present invention. For example, a compartmentalized kit in
accordance with the present invention includes any kit in which
reagents are contained in separate containers. Such containers
include small glass containers, plastic containers or strips of
plastic or paper. Such containers allow the efficient transfer of
reagents from one compartment to another compartment such that the
samples and reagents are not cross-contaminated and the agents or
solutions of each container can be added in a quantitative fashion
from one compartment to another. Such containers will include a
container which will accept the test sample (DNA protein or cells),
a container which contains the primers used in the assay,
containers which contain enzymes, containers which contain wash
reagents, and containers which contain the reagents used to detect
the extension products.
[0096] The present invention also relates to a kit comprising the
oligonucleotide primer of the present invention, which are specific
to either one of the PCA3 mRNA lacking the additional sequence of
the present invention or the PCA3 mRNA containing the additional
sequence of the present invention.
[0097] Further objects and advantages of the present invention will
be clear from the description that follows.
BRIEF DESCRIPTION OF THE DRAWINGS
[0098] Having thus generally described the invention, reference
will now be made to the accompanying drawings, showing by way of
illustration a preferred embodiment thereof, and in which:
[0099] FIG. 1 shows the PCA3 genomic structure and location of
oligonucleotides used for PCR;
[0100] FIG. 2 shows a gel separating PCA3 RT-PCR products amplified
from tissue biopsies of prostate cancer and benign prostate
hyperplasia, using the primers of Example 1;
[0101] FIG. 3 illustrates the nucleic acid sequences of
RT-PCR-amplified PCA3 fragments with and without the additional
sequence of the present invention. The sequences were amplififed
using PCR primers located in exon 3 and exon 4a. Primer sequences
are shown in bold letters. Capital letters represent nucleic acids
common to both sequences;
[0102] FIG. 4 shows the amino acid sequence predicted from the PCA3
mRNAs containing the additional sequence of the present invention.
This sequence corresponds to amino acids 1-23 of the original PCA3
polypeptide; and
[0103] FIG. 5 shows examples of antigenic epitope-bearing PCA3
peptides comprising 8 amino acids (calculated according to H. G.
Rammensee et al., 1995, MHC ligands and peptide motifs: first
listing, in Immunogenics; 41(4)). The SEQ ID NOs of the exemplified
antigenic epitopes are indicated on the right (SEQ ID NOs 5 to
12).
[0104] Other objects, advantages and features of the present
invention will become more apparent upon reading of the following
non-restrictive description of preferred embodiments with reference
to the accompanying drawing which is exemplary and should not be
interpreted as limiting the scope of the present invention.
DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS
[0105] In one embodiment, the present invention relates to an
isolated and/or purified differentially-expressed PCA3 mRNA
molecule. Preferably, the PCA3 mRNA or nucleic acid molecule
comprises a polynucleotide sequence at least 90% identical (more
preferably, 95%, 96%, 97%, 98%, 99% or 100% identical) to a
sequence selected from the group consisting of:
(a) a nucleotide sequence encoding a differentially expressed PCA3
polypeptide comprising the complete amino acid sequence in SEQ ID
NO: 3; (b) a nucleotide sequence complementary to any of the
nucleotide sequences in (a) or (b).
[0106] In one preferred embodiment, the isolated nucleic acid
molecule comprises a differentially expressed PCA3 mRNA nucleotide
sequence with greater than 90% identity or similarity to the
nucleotide sequence present in SEQ ID NO: 1 (preferably greater
than 95%, 96%, 97%, 98%, 99% or 100%). In another preferred
embodiment, the isolated nucleic acid molecule comprises the
differentially expressed PCA3 mRNA sequence lacking the additional
sequence present in SEQ ID NO:2. In another embodiment, the
isolated differentially expressed additional sequence-minus mRNA
sequence nucleic acid molecule encodes the differentially expressed
PCA3 amino acid sequence present in SEQ ID NO:3.
[0107] Although PCT application CA98/00346 teaches a number of
alternatively spliced mRNAs, prior to the present invention, a PCA3
mRNA comprising an additional sequence between exon 3 and exon 4a
had not been identified. Furthermore, the identification of this
additional sequence as a distinguishing marker of the prostate
state had not been made. In addition, the correlation between the
PCA3 mRNA minus the additional sequence and prostate cancer (as
opposed to the PCA3 mRNA-containing the additional sequence in
non-prostate cancer [i.e. normal or BPH]) had not been made. Thus,
the additional sequence in PCA3 mRNA enables a prognosis and
diagnosis of prostatic diseases in a patient. Preferably, the PCA3
nucleic acid molecule comprises a polynucleotide sequence at least
90% identical (more preferably, 95%, 96%, 97%, 98%, 99% or 100%
identical) to one of the above-described differentially expressed
mRNAs.
[0108] Also included within the scope of this invention are the
functional equivalents of the herein-described isolated nucleic
acid molecules and derivatives thereof. For example, the nucleic
acid sequences depicted in SEQ ID NO:1 and SEQ ID NO:2 can be
altered by substitutions, additions or deletions that provide for
functionally equivalent molecules. Due to the degeneracy of
nucleotide coding sequences, other DNA sequences which encode
substantially the same amino acid sequence as depicted in SEQ ID
NO: 3 can be used in the practice of the present invention.
[0109] In addition, the nucleic acid sequence can comprise a
nucleotide sequence which results from the addition, deletion or
substitution of at least one nucleotide to the 5'-end and/or the
3'-end of the nucleic acid formula shown in SEQ ID NO:1 or 2 or a
derivative thereof. Any nucleotide or polynucleotide can be used in
this regard, provided that its addition, deletion or substitution
does not substantially alter the amino acid sequence of SEQ ID NO:3
which is encoded by the additional sequence-containing nucleotide
sequence. Moreover, the nucleic acid molecule of the present
invention can, as necessary, have restriction endonuclease
recognition sites added to its 5-end and/or 3'-end. All variations
of the nucleotide sequence of the PCA3 nucleotide coding sequence
and fragments thereof permitted by the genetic code are, therefore,
included in this invention.
[0110] Further, it is possible to delete codons or to substitute
one or more codons by codons other than degenerate codons to
produce a structurally modified polypeptide, but one which has
substantially the same utility or activity of the polypeptide
produced by the unmodified nucleic acid molecule. As recognized in
the art, the two polypeptides are functionally equivalent, as are
the two nucleic acid molecules which give rise to their production,
even though the differences between the nucleic acid molecules are
not related to degeneracy of the genetic code.
[0111] One skilled in the art will realize that genomes often
contain slight allelic variations between individuals. Therefore,
the isolated nucleic acid molecule is also intended to include
allelic variations, so long as the sequence is a functional
derivative of the differentially expressed PCA3 mRNA coding
sequence. When a PCA3 allele does not encode the identical sequence
to that found in SEQ ID Nos:1 or 2, it can be isolated and
identified as PCA3 using the same techniques used herein, and
especially PCR techniques to amplify the appropriate gene with
primers based on the sequences disclosed herein.
[0112] One skilled in the art will realize that organisms other
than humans might also contain differentially-expressed PCA3 mRNAs
(for example, eukaryotes; more specifically, mammals, birds, fish,
and plants; more specifically, gorillas, rhesus monkeys, and
chimpanzees). The invention is intended to include, but not be
limited to, differentially-expressed PCA3 mRNAs isolated from the
above-described organisms.
[0113] Isolated nucleic acid molecules of the present invention are
also meant to include those chemically synthesized. For example, a
nucleic acid molecule with the nucleotide sequence described herein
or encoding the herein described differentially expressed products
of PCA3 gene can be designed and, if necessary, divided into
appropriate smaller fragments. Then an oligomer which corresponds
to the nucleic acid molecule, or to each of the divided fragments,
can be synthesized. Such synthetic oligonucleotides can be
prepared, for example, by the triester method of Matteucci et al.,
J. Am. Chem. Soc. 103:3185-3191 (1981) or by using an automated DNA
synthesizer.
[0114] In another embodiment, the present invention relates to
purified differentially expressed polypeptides (preferably,
substantially pure) having an amino acid sequence corresponding to
the herein described PCA3, or a functional derivative thereof. In a
preferred embodiment, the polypeptide has the amino acid sequence
set forth in SEQ ID NO: 3 or mutant or species variation thereof,
or at least 80% identity or at least 90% similarity thereof
(preferably, at least 90%, 95%, 96%, 97%, 98%, or 99% identity or
at least 95%, 96%, 97%, 98%, or 99% similarity thereof), or at
least 6 contiguous amino acids thereof (preferably, at least 10,
15, 20, 25, or 50 contiguous amino acids thereof).
[0115] In a preferred embodiment, the invention relates to
differentially-expressed PCA3 epitopes. The epitope of these
polypeptides is an immunogenic or antigenic epitope. An immunogenic
epitope is that part of the protein which elicits an antibody
response when the whole protein is the immunogen. An antigenic
epitope is a fragment of the protein which can elicit an antibody
response. Methods of selecting antigenic epitope fragments are well
known in the art. See, Sutcliffe et al., Science 219:660-666
(1983). Antigenic epitope-bearing peptides and polypeptides of the
invention are useful to raise an immune response that specifically
recognizes the polypeptides. Antigenic epitope-bearing peptides and
polypeptides of the invention comprise at least 7 amino acids
(preferably, 9, 10, 12, 15 or 20 amino acids) of the proteins of
the invention. Examples of an antigenic peptide are shown in FIG. 5
as predicted using the method of Rammensee et al., (1995, MHC
ligands and peptide motifs: first listing, in Immunogenics; 41(4)).
Of course, it will be realized that other epitope-bearing PCA3
peptides could be predicted and used to raise antibodies.
[0116] A variety of methodologies known in the art can be utilized
to obtain the peptide of the present invention. In one embodiment,
the peptide is purified from tissues or cells which naturally
produce the peptide. Alternatively, the above-described isolated
nucleic acid fragments can be used to express the differentially
expressed PCA3 protein in any organism. The samples of the present
invention include cells, protein extracts or membrane extracts of
cells, or biological fluids. The sample will vary based on the
assay format, the detection method and the nature of the tissues,
cells or extracts used as the sample.
[0117] Any organism can be used as a source for the peptide of the
invention, as long as the source organism naturally contains such a
peptide. As used herein, "source organism" refers to the original
organism from which the amino acid sequence of the subunit is
derived, regardless of the organism the subunit is expressed in and
ultimately isolated from.
[0118] In another embodiment, the present invention relates to a
nucleic acid for the specific detection of the presence of PCA3
nucleic acid in a sample comprising the above-described nucleic
acid molecules or at least a fragment thereof which binds under
stringent conditions to PCA3 nucleic acid.
[0119] In one prefered embodiment, the present invention relates to
nucleic acid probes which are complementary to a nucleotide
sequence consisting of at least 10 consecutive nucleotides
(preferably, 15, 18, 20, 25, or 30) from the nucleic acid molecule
comprising a polynucleotide sequence at least 90% identical to a
sequence selected from the group consisting of:
(a) a nucleotide sequence encoding the PCA3 polypeptide comprising
the complete amino acid sequence in SEQ ID NO: 3; (b) a nucleotide
sequence encoding the PCA3 gene comprising the nucleotide sequence
in SEQ ID NO: 1, or 2; (c) a nucleotide sequence complementary to
any of the nucleotide sequences in (a) or (b), and (d) a nucleotide
sequence as previously described above.
[0120] In one embodiment of the above described method, a nucleic
acid probe is immobilized on a solid support. Examples of such
solid supports include, but are not limited to, plastics such as
polycarbonate, complex carbohydrates such as agarose and sepharose,
and acrylic resins, such as polyacrylamide and latex beads.
Techniques for coupling nucleic acid probes to such solid supports
are well known in the art.
[0121] The test samples suitable for nucleic acid probing methods
of the present invention include, for example, cells or nucleic
acid extracts of cells, or biological fluids. The sample used in
the above-described methods will vary based on the assay format,
the detection method and the nature of the tissues, cells or
extracts to be assayed. Methods for preparing nucleic acid extracts
of cells are well known in the art and can be readily adapted in
order to obtain a sample which is compatible with the method
utilized.
[0122] In another embodiment, the present invention relates to a
method of detecting the presence of differentially expressed PCA3
mRNA in a sample comprising : a) contacting the sample with the
above-described nucleic acid probe, under specific hybridization
conditions such that hybridization occurs, and b) detecting the
presence of the probe bound to the nucleic acid molecule. One
skilled in the art would select the nucleic acid probe according to
techniques known in the art as described above. Samples to be
tested include but should not be limited to RNA samples from human
tissue.
[0123] Having identified that the additional PCA3 sequence of the
present invention can be used as a marker for distinguishing
between malignant and non malignant prostate states, probes which
are specific to this additional sequence (or variants or fragments
thereof) could also be used in accordance with the present
invention. Of course, since in certain embodiments, such probes
might detect genomic DNA, a positive signal coming from the genomic
DNA might have to be eliminated in order to specifically detect the
differentially expressed PCA3 mRNA.
[0124] Although the present invention is specifically demonstrated
using primers hybridizing to exon 3 and exon 4a sequences, it
should be clear to the skilled artisan that primers derived from
other regions of PCA3 could be used. For example, primers could be
derived from sequences of exon 2, exon 4b, exon 4c or to the
additional sequence thereof. Methods to derive specific primers
from known sequences are well known in the art.
[0125] In another embodiment, the present invention relates to a
kit for detecting the presence of differentially expressed PCA3
mRNA in a sample comprising at least one container means having
disposed therein the above-described nucleic acid probe. In a
preferred embodiment, the kit further comprises other containers
comprising one or more of the following: wash reagents and reagents
capable of detecting the presence of bound nucleic acid probe.
Examples of detection reagents include, but are not limited to
radiolabelled probes, enzymatic labeled probes (horse radish
peroxidase, alkaline phosphatase), and affinity labeled probes
(biotin, avidin, or steptavidin).
[0126] In more detail, a compartmentalized kit includes any kit in
which reagents are contained in separate containers. Such
containers include small glass containers, plastic containers or
strips of plastic or paper. Such containers allow the efficient
transfer of reagents from one compartment to another compartment
such that the samples and reagents are not cross-contaminated and
the agents or solutions of each container can be added in a
quantitative fashion from one compartment to another. Such
containers will include a container which will accept the test
sample, a container which contains the probe or primers used in the
assay, containers which contain wash reagents (such as phosphate
buffered saline, Tris-buffers, and the like), and containers which
contain the reagents used to detect the hybridized probe, bound
antibody, amplified product, or the like.
[0127] One skilled in the art will readily recognize that the
nucleic acid probes described in the present invention can readily
be incorporated into one of the established kit formats which are
well known in the art.
[0128] In another embodiment of the present invention (and
similarly to probes of the present invention) the antibodies of the
present invention can be immobilized on a solid support. Examples
of such solid supports include plastics such as polycarbonate,
complex carbohydrates such as agarose and sepharose, acrylic resins
and such as polyacrylamide and latex beads. Techniques for coupling
antibodies to such solid supports are well known in the art. The
immobilized antibodies of the present invention can be used for in
vitro, in vivo, and in situ assays as well as in
immunochromatography.
[0129] In another embodiment, the present invention relates to a
method of detecting a differentially expressed PCA3 polypeptide in
a sample, comprising: a) contacting the sample with an
above-described antibody (or protein), under conditions such that
immunocomplexes form, and b) detecting the presence of the antibody
bound to the polypeptide. In detail, the methods comprise
incubating a test sample with one or more of the antibodies of the
present invention and assaying whether the antibody binds to the
test sample. The relative levels of differentially expressed PCA3
in a sample enable a distiction between a malignant and
non-malignant prostatic state.
[0130] In a further embodiment, the present invention relates to a
method of detecting a PCA3 antibody in a sample, comprising: a)
contacting the sample with an above-described differentially
expressed PCA3 protein, under conditions such that immunocomplexes
form, and b) detecting the presence of the protein bound to the
antibody or antibody bound to the protein. In detail, the methods
comprise incubating a test sample with one or more of the proteins
of the present invention and assaying whether the antibody binds to
the test sample.
[0131] In another embodiment of the present invention, a kit is
provided which contains all the necessary reagents to carry out the
previously described methods of detection.
[0132] The kit can comprise: i) a first container means containing
an above-described antibody, and ii) second container means
containing a conjugate comprising a binding partner of the antibody
and a label.
[0133] The kit can also comprise: i) a first container means
containing an above-described protein, and preferably, ii) second
container means containing a conjugate comprising a binding partner
of the protein and a label. More specifically, a diagnostic kit
comprises a differentially expressed PCA3 protein as described
above, to detect antibodies in the serum of potentially infected
animals or humans.
[0134] In another preferred embodiment, the kit further comprises
one or more other containers comprising one or more of the
following: wash reagents and reagents capable of detecting the
presence of bound antibodies. Examples of detection reagents
include, but are not limited to, labeled secondary antibodies, or
in the alternative, if the primary antibody is labeled, the
chromophoric, enzymatic, or antibody binding reagents which are
capable of reacting with the labeled antibody. The
compartmentalized kit can be as described above for nucleic acid
probe kits.
[0135] One skilled in the art will readily recognize that the
antibodies described in the present invention can readily be
incorporated into one of the established kit formats which are well
known in the art.
[0136] It is to be understood that although the following
discussion is specifically directed to human patients, the
teachings are also applicable to any animal that differentially
expresses PCA3 mRNAs.
[0137] The diagnostic and screening methods of the invention are
especially useful for a patient suspected of being at risk for
developing a disease associated with an altered expression level of
PCA3 based on family history, or a patient in which it is desired
to diagnose a PCA3-related disease (ex. prostate cancer).
[0138] According to the invention, presymptomatic screening of an
individual in need of such screening is now possible using DNA
encoding the PCA3 protein or the PCA3 gene of the invention or
fragments thereof. The screening method of the invention allows a
presymptomatic diagnosis, of the presence of the PCA3-minus
additional sequence, differentially expressed PCA3 mRNA in
individuals, and thus an opinion concerning the likelihood that
such individuals would develop or have developed a PCA3-associated
disease or have a normal prostatic state. This is especially
valuable for the identification of carriers of altered PCA3 genes,
for example, from individuals with a family history of a
PCA3-associated disease. Early diagnosis is also desired to
maximize appropriate timely intervention.
[0139] In one preferred embodiment of the method of screening, a
tissue sample would be taken from such individual, and screened for
(1) the presence of the PCA3 mRNA lacking the additional sequence
of the present invention; (2) the presence of the additional
sequence-containing PCA3 mRNA and/or (3) the presence of
differentially expressed PCA3 protein. PCA3 mRNA can be
characterized and compared to determine differentially expressed
PCA3 mRNA (a) levels and/or (b) size. Lastly, differentially
expressed PCA3 protein can be (a) detected and/or (b) quantitated
using a biological assay for PCA3 activity or using an
immunological assay and PCA3 antibodies A presence of a PCA3 mRNA
lacking the additional sequence (or an mRNA not modifying and/or
not interrupting the PCA3 coding sequence) and/or of the protein
encoded thereby would indicate that the patient is at risk for
developing prostate cancer, or has developped prostate cancer. A
presence of a PCA3 mRNA containing the additional sequence of the
present invention and/or of the protein encoded thereby, in the
absence of PCA3 mRNA lacking the additional sequence and/or the
protein encoded thereby or at a level superior to that of the mRNA
lacking the additional sequence and/or the protein encoded thereby
would indicate that the patient has not yet developed prostate
cancer, and/or has a lower risk of developing prostate cancer.
[0140] Therapeutic effects of therapeutic nucleic acids can
include, but are not limited to turning off or modifying the
processing of the differentially expressed PCA3 mRNA lacking the
additional sequence of the present invention. In addition, an
expression of a differentially-expressed PCA3 mRNA comprising the
additional sequence in accordance with the present invention to a
higher level than that of the PCA3 mRNA lacking the additional
sequence could have cancer-reversing effects on cells.
[0141] Included as well in the invention are pharmaceutical
compositions comprising an effective amount of at least one
antisense oligonucleotide to a PCA3 mRNA lacking the additional
sequence, in combination with a pharmaceutically acceptable
carrier. Such antisense oligos include, but are not limited to, at
least one nucleotide sequence of 12-500 bases in length which is
complementary to at least a portion of SEQ ID NO:2.
[0142] Thus, broadly, the invention provides means to shift the
balance between the quantity of the differentially expressed PCA3
mRNAs such that the malignant state of a cell can be modulated.
[0143] Specificity for gene expression in prostate cancer cells can
be conferred by using appropriate cell-specific regulatory
sequences, such as cell-specific enhancers and promoters. Thus,
gene therapy can be used to alleviate PCA3 related pathology by
inhibiting the inappropriate expression of a particular form of
PCA3. Moreover, gene therapy can be used to alleviate such
pathologies by providing the appropriate expression level of a
particular form of PCA3. In this case, particular PCA3 nucleic acid
sequences can be coded by DNA or RNA constructs which are
administered in the form of viruses, as described above.
[0144] The present invention provides the above-described PCA3
antibodies (preferably, PCA3 murine antibodies and chimeric PCA3
murine-human antibodies, and fragments and regions thereof) which
inhibit or neutralize PCA3 biological activity in vivo and are
specific for PCA3. These antibodies can be used for therapeutic
purposes in subjects having pathologies or conditions associated
with the presence of aberrant PCA3 expression. Antibodies, and
fragments, regions and derivatives thereof, of the present
invention preferably contain at least one region which recognizes
an epitope of PCA3 which has inhibiting and/or neutralizing
biological activity in vivo.
[0145] Treatment comprises parenterally administering a single or
multiple doses of the antibody, fragment or derivative. Preferred
for human pharmaceutical use are high affinity potent
PCA3-inhibiting and/or neutralizing murine and chimeric antibodies,
fragments and regions of this invention.
[0146] Monoclonal antibodies of the present invention may be
administered by any means that enables the active agent to reach
the agent's site of action in the body of a mammal. Because
proteins are subject to being digested when administered orally,
parenteral administration, i.e., intravenous, subcutaneous,
intramuscular, would ordinarily be used to optimize absorption.
[0147] Monoclonal antibodies of the present invention may be
administered either as individual therapeutic agents or in
combination with other therapeutic agents. They can be administered
alone, but are generally administered with a pharmaceutical carrier
selected on the basis of the chosen route of administration and
standard pharmaceutical practice.
[0148] The dosage administered will, of course, vary depending upon
known factors such as the pharmacodynamic characteristics of the
particular agent, and its mode and route of administration; age,
health, and weight of the recipient; nature and extent of symptoms,
kind of concurrent treatment, frequency of treatment, and the
effect desired. Usually a daily dosage of active ingredient can be
about 0.1 to 100 milligrams per kilogram of body weight. Ordinarily
0.5 to 50, and preferably 1 to 10 milligrams per kilogram per day
given in divided doses 1 to 6 times a day or in sustained release
form is effective to obtain desired results.
[0149] Dosage forms (composition) suitable for internal
administration generally contain from about 1 milligram to about
500 milligrams of active ingredient per unit. In these
pharmaceutical compositions the active ingredient will ordinarily
be present in an amount of about 0.5-95% by weight based on the
total weight of the composition.
[0150] Cytotoxic drugs which can be conjugated to antibodies and
subsequently used for in vivo therapy include, but are not limited
to, daunorubicin, doxorubicin, methotrexate, and Mitomycin C.
[0151] The non-human animals of the invention comprise any animal
having a transgenic interruption or alteration of the endogenous
gene(s) (knock-out animals) and/or into the genome of which has
been introduced one or more transgenes that direct the expression
of diffrentially expressed human PCA3 mRNAs. Also prefered are the
introduction of antisense PCA3 nucleic acids.
[0152] Such non-human animals include vertebrates such as rodents,
non-human primates, sheep, dog, cow, amphibians, reptiles, etc.
Preferred non-human animals are selected from non-human mammalian
species of animals, most preferably, animals from the rodent family
including rats and mice, most preferably mice.
[0153] The transgenic animals of the invention are animals into
which has been introduced by nonnatural means (i.e., by human
manipulation), one or more genes that do not occur naturally in the
animal, e.g., foreign genes, genetically engineered endogenous
genes, etc. The nonnaturally introduced genes, known as transgenes,
may be from the same or a different species as the animal but not
naturally found in the animal in the configuration and/or at the
chromosomal locus conferred by the transgene. Transgenes may
comprise foreign DNA sequences, i.e., sequences not normally found
in the genome of the host animal. Alternatively or additionally,
transgenes may comprise endogenous DNA sequences that are abnormal
in that they have been rearranged or mutated in vitro in order to
alter the normal in vivo pattern of expression of the gene, or to
alter or eliminate the biological activity of an endogenous gene
product encoded by the gene.
[0154] The transgenic non-human animals of the invention are
produced by introducing transgenes into the germline of the
non-human animal. Embryonic target cells at various developmental
stages are used to introduce the transgenes of the invention.
Different methods are used depending on the stage of development of
the embryonic target cell(s). These methods are well known in the
art.
[0155] Transgenes may be introduced into non-human animals in order
to provide animal models for human diseases. Transgenes that result
in such animal models include, e.g., transgenes that encode the
differentially expressed PCA3 mRNAs associated with a malignant
prostate status (i.e., prostate cancer) ora non-malignant prostate
status.
[0156] Having identified a marker sequence in differentially
expressed PCA3 mRNA and a correlation between the balance of the
expression level of the differentially expressed PCA3 mRNAs (or
protein encoded thereby) and the malignant or non-malignant
prostatic states, the present invention opens the way to numerous
methods, assays and reagents for the prognosis, diagnosis, staging,
predisposition and therapy of prostate cancer. In a broad
embodiment, the present invention provides the means to assess
prostate cancer by identifying PCA3 mRNA lacking the additional
sequence in accordance with the present invention (or a protein
encoded thereby). Numerous methods, primers, probes, antibodies and
reagents can be used to identify such a nucleic acid molecule (or
such a protein), as will be clear to the skilled artisan to which
the present invention pertains.
[0157] The present invention is illustrated in further detail by
the following non-limiting examples.
EXAMPLE 1
Identification of Differentially Expressed PCA3 mRNAs and
Correlation of Their Expression with Prostatic Disease
[0158] PCA3-specific PCR primers were developed in order to analyze
PCA3 expression in different samples. To be able to discern between
sequences amplified from mRNA (messenger RNA) and genomic DNA,
these primers were designed to span an intron, in occurence intron
3. As illustrated in FIG. 1, the PCA3 sense primer lies within exon
3 and the PCA3 antisense primer within exon 4a. Samples to be
analyzed for PCA3 expression consisted of frozen tissue chips
removed by transurethral resection of the prostate (BPH, 4
patients) or frozen prostates obtained by radical prostatectomy
(prostate cancer, 6 patients). Radical prostatectomy samples were
processed into frozen sections to specifically select regions
containing prostate cancer cells. RNA was extracted from the frozen
samples using a liquid-phase RNA extraction method (Trizol.TM.).
Extracted nucleic acids were subsequently treated with DNase in
order to digest genomic DNA. DNase-treated RNA was reverse
transcribed into cDNA using reverse transcriptase and then
submitted to PCR-analysis using the PCA3 primers. PCR was performed
for 35 cycles with Taq DNA polymerase, amplified material was
separated on agarose gels and visualized by ethidium bromide
staining. As shown in FIG. 2, PCR amplification of PCA3 generates
two products which can be separated by size and differ in relative
abundancy. The smaller amplicon (277 bp) is predominantly or
exclusively found in samples from prostate cancer patients (FIG. 2,
upper row) whereas the larger amplicon (505 bp) is more prominent
in samples from patients with a non-malignant prostatic state (BPH
[FIG. 2, lower row]). Pathological examination of patients'
biopsies confirmed the initial diagnosis for each patient except
for Patient BPH 1 which was found to have prostate cancer.
[0159] In order to confirm the origin of the amplified fragments,
they were isolated from the gel and sequenced. Sequences are shown
in FIG. 3. As expected, the smaller 277 by fragment proved to
correspond to the regions of exons 3 and 4a spanned by the PCA3 PCR
primers. The larger 505 by fragment is identical to the smaller
fragment except for the herein identified sequence which lies
between exon 3 and exon 4a. Of note, direct PCR analysis of all
samples without reverse transcription did not yield amplified
material ruling out the hypothesis that the larger amplification
product originates from genomic DNA.
[0160] Thus, PCA3 mRNA is present in at least two distinct forms
within the cell, a short form lacking the herein identified
additional sequence (hereafter called sequence 2; SEQ ID NO:2) as
well as a long form having this additional sequence (hereafter
called sequence 1; SEQ ID NO:1). The presence of the additional
sequence in sequence 1 interrupts the predicted open reading frame
coding for the PCA3 protein. The predicted sequence of the protein
encoded by this long PCA3 mRNA is shown in FIG. 4. As illustrated
in FIG. 2, relative expression levels of the two PCA3 mRNA
sequences vary dependent on the cell type. Prostate cancer cells
predominantly express sequence 2 whereas BPH cells mainly express
sequence 1.
[0161] These observations demonstrate that it is possible to
discern between a malignant and non-malignant state of a prostate.
As well, it is tempting to predict that the relative levels of the
two types of PCA3 mRNAs, will enable to discern the benign state
from the malignant state.
EXAMPLE 2
Assessment of the Prostatic State of a Patient Using RT-PCR
[0162] Patient samples were obtained and RNA prepared therefrom as
commonly known. Reverse transcription mixes were prepared as RT
follows: 0.2 pg total RNA+0.6 .mu.g pdN6 (random hexamer
primers)+1.25 mM dNTPs+200 U M-MLV reverse transcriptase in 50 mM
Tris-HCl pH 8.3, 75 mM KCl, 3 mM MgCl.sub.2, 10 mM DTT. The mixture
was incubated 1 hr at 40.degree. C.
[0163] 4 .mu.l of the RT-reaction of above was mixed in 50 .mu.L of
20 mM Tris-HCl pH 8.4, 50 mM KCl, 2.5 mM MgCl.sub.2, 0.5 mM dNTPs,
0.5 .mu.M of each primer and 2.5 U Taq DNA polymerase. For PCR
analysis, the amplification was carried out for 35 cycles (1 min
each at 94.degree. C., 60.degree. C., 72.degree. C.) followed by a
10 min extension at 72.degree. C. The PCR products were analyzed by
conventional agarose gel electrophoresis.
[0164] Although the present invention has been described
hereinabove by way of preferred embodiments thereof, it can be
modified, without departing from the spirit and nature of the
subject invention as defined in the appended claims.
Sequence CWU 1
1
121506DNAHomo sapiens 1caggaagcac aaaaggaagc acagaggtaa gtgctttata
aagcactcaa tttctactca 60gaaatttttg atggccttaa gttcctctac tcgtttctat
ccttcctact cactgtcctc 120ccggaatcca ctaccgattt tctatttctt
gcctcgtatt gtctgactgg ctcacttgga 180tttatcctca cggagtctgg
attttctacc cgggctcacc tccgtccctc catatttgtc 240ctccactttc
acagatccct gggagaaatg cccggccgcc atcttgggtc atcgatgagc
300ctcgccctgt gcctggtccc gcttgtgagg gaaggacatt agaaaatgaa
ttgatgtgtt 360ccttaaagga tgggcaggaa aacagatcct gttgtggata
tttatttgaa cgggattaca 420gatttgaaat gaagtcacca aagtgagcat
taccaatgag aggaaaacag acgagaaaat 480cttgatggct tcacaagaca tgcaac
5062278DNAHomo sapiens 2caggaagcac aaaaggaagc acagagatcc ctgggagaaa
tgcccggccg ccatcttggg 60tcatcgatga gcctcgccct gtgcctggtc ccgcttgtga
gggaaggaca ttagaaaatg 120aattgatgtg ttccttaaag gatgggcagg
aaaacagatc ctgttgtgga tatttatttg 180aacgggatta cagatttgaa
atgaagtcac caaagtgagc attaccaatg agaggaaaac 240agacgagaaa
atcttgatgg cttcacaaga catgcaac 278323PRTHomo sapiens 3Met Phe Leu
His Ile Ser Ser Pro Phe Lys Tyr Pro His Thr Gln Glu1 5 10 15Ala Gln
Lys Glu Ala Gln Arg 20420DNAArtificial SequenceDescription of
Artificial Sequence oligonucleotide 4gagtaggaag gatagaaacg
2058PRTArtificial SequenceDescription of Artificial Sequence PCA3
epitope 5Leu Ala Leu Cys Leu Val Pro Leu1 568PRTArtificial
SequenceDescription of Artificial Sequence PCA3 epitope 6Gly Glu
Met Pro Gly Arg His Leu1 578PRTArtificial SequenceDescription of
Artificial Sequence PCA3 epitope 7Leu Gly Ser Ser Met Ser Leu Ala1
588PRTArtificial SequenceDescription of Artificial Sequence PCA3
epitope 8Val Pro Leu Val Arg Glu Gly His1 598PRTArtificial
SequenceDescription of Artificial Sequence PCA3 epitope 9Ser Pro
Phe Lys Tyr Pro His Thr1 5108PRTArtificial SequenceDescription of
Artificial Sequence PCA3 epitope 10Gln Glu Ala Gln Lys Glu Ala Gln1
5118PRTArtificial SequenceDescription of Artificial Sequence PCA3
epitope 11Thr Gln Glu Ala Gln Lys Glu Ala1 5128PRTArtificial
SequenceDescription of Artificial Sequence PCA3 epitope 12Phe Leu
His Ile Ser Ser Pro Phe1 5
* * * * *